title: Technical advance: actin CytoFRET, a novel FRET flow cytometry method for detection of actin dynamics in resting and activated T cell.
abstract: Actin cytoskeleton plays a critical role in regulating T cell motility and activation. However, the lack of a real-time quantitative method to analyze actin assembly has limited the progress toward understanding actin regulation. Here, we describe a novel approach to probe actin dynamics on living T cells using FRET combined with flow cytometry. We have first generated a Jurkat T cell line stably coexpressing EGFP and mOrange FPs fused to actin. The real-time variation of actin monomer assembly or disassembly into filaments was quantified using a ratiometric flow cytometry method measuring changes in the mOrange/EGFP emission ratio. The method was validated on resting T cells by using chemical compounds with known effects on actin filaments and comparison with conventional microscopy imaging. Our method also detected the rapid and transient actin assembly in T cells stimulated by anti-CD3/CD28-coated beads, demonstrating its robustness and high sensitivity. Finally, we provide evidence that lentiviral-mediated transduction of shRNAs in engineered Jurkat cells could be used as a strategy to identify regulators of actin remodeling. In conclusion, the flow cytometric FRET analysis of actin polymerization represents a new technical advance to study the dynamics of actin regulation in intact cells.
authors.affiliation: Tolérance Immunitaire, Université de Nice Sophia-Antipolis, Nice, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Larbret
authors.forename: Nicholas
authors.initials: N
authors.lastname: Dubois
authors.forename: Frédéric
authors.initials: F
authors.lastname: Brau
authors.forename: Elodie
authors.initials: E
authors.lastname: Guillemot
authors.forename: Karim
authors.initials: K
authors.lastname: Mahiddine
authors.forename: Sophie
authors.initials: S
authors.lastname: Tartare-Deckert
authors.forename: Valérie
authors.initials: V
authors.lastname: Verhasselt
authors.forename: Marcel
authors.initials: M
authors.lastname: Deckert
mesh_terms: D000199:Actins / Q000032:analysis*; D018106:CD28 Antigens / Q000502:physiology; D017252:CD3 Complex / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000737:chemistry*; D051316:Wiskott-Aldrich Syndrome Protein
chemical_list: D000199:Actins; D018106:CD28 Antigens; D017252:CD3 Complex; D051316:Wiskott-Aldrich Syndrome Protein
keywords: FRET; Flow cytometry; actin; fluorescent protein